Cytoprotective fatty moiety compounds

ABSTRACT

Water or lipid soluble, pharmacologically active, antioxidant, anti-phospholipase compounds that are chemically defined. The compounds protect mammalian cells by inhibiting PLA 2  and preventing oxidation. In particular, each compound has at least two fatty moieties and no active hydroxy group. The compound may also have at least one ionizable group, which may be a carboxyl group, and each of the fatty moieties has from sixteen to twenty carbon atoms and at least one cis-unsaturated double bond.

CROSS-REFERENCES TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/467,690, filed Jun. 6,1995, now U.S. Pat. No. 5,659,049, which is a division of abandonedapplication Ser. No. 08/010,456, filed Jan. 27, 1993, now abandoned,which is a continuation of abandoned application Ser. No. 07/839,780,filed Apr. 1, 1992, now abandoned, which is a continuation-in-part ofprior application Ser. No. 07/399,941 filed Aug. 29, 1989, nowabandoned, which in turn is a continuation-in-part of prior applicationSer. No. 07/256,330 filed Oct. 11, 1988, now abandoned, which again inturn is a continuation-in-part of prior application Ser. No. 07/156,739filed Feb. 18, 1988, now abandoned and Ser. No. PCT/US87/00408 filedFeb. 24, 1987, refiled as application Ser. No. 07/653,483, nowabandoned. This application and the identified prior applications areall assigned to the common assignee, Virginia Commonwealth University,and the subject matter of each prior application is hereby specificallyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and materials for protectingmammalian cells from injury due to intrinsic membrane lysis, oxidationand/or invasion by destructive agents. In particular the inventionrelates to materials and methods for treating against and/orprophylactically inhibiting the injury causation. Even moreparticularly, the invention relates to bioactive agents and the usethereof for treating or prophylactically inhibiting phospholipasemediated injury and/or injury due to oxidation. In a specific sense theinvention provides agents for preventing and/or treating inflammationand cell destruction in mammalian tissue and for protection andpreservation of biologic material such as food and tissue samples.

2. The Prior Art Environment

The base structure of all living organisms is the cell which isstructurally defined by its membranous lipoprotein envelope. Themembranous network that holds the cell together maintains the ionicbalance and provides the receptors to hormones and neurotransmittersthat enable a cell to interact with its environment. This is pertinentto interaction with neighboring cells which enable isolated cells,tissues, or whole organisms to survive as both independent units and asparticipants in cellular interactions, in vitro and in vivo.Nutritional, kinetic, electro-physiological, excretory, and reproductivemechanisms are mediated through the self renewal of the lipoproteinmembranes that bind the cell, its nucleus and organelles into afunctional whole.

The cell has a preordained life to live in accordance with the balancesuperimposed by the information provided by the nucleus and theenvironment. A cell has a date of conception and a circumstance mediatedor preordained time to die. The dictates of certain circumstances, i.e.,physiological stimuli or pathologic injury, prescribe the manner ofdeath and the time of death of cells. Cell death and/or injury is theresult of both intrinsic and extrinsic factors and is key to the fate ofthe larger organism of which cells are a critical and necessary part.

That which is good for the cell, that which maintains its capacity torespond to change to ionic fluxes that maintain membrane potential andto repair injury under normal conditions should be good for its host orsupportive to in vitro biologic production (i.e., tissue culture,monoclonal antibody, enzyme or endocrine production of pertinence tobiotechnology). The integrity of cell membranes which maintain ionicflux, and electro-physiologic and/or hormonal or messenger responses isthe key to cellular functional survival and longevity. Repair andresistance to injury is a function of the maintenance of lipoproteinmembrane integrity.

Factors which govern cell function, renewal, reproduction and death arecontrolled by their effects on the phospholipid/protein envelope orcytoskeleton. The cell membrane controls the cellular clocks and ionicfluxes which govern responsiveness and survival. Damage tophospholipid/proteins, with particular emphasis on lipid peroxidation,membrane oxidation and the action of phospholipases, governs resistanceto injury, repair and host responses to environmental change and ionicand osmotic integrity.

Pathological events in a host under clinical circumstances result inmassive cellular insult, initiated or mediated by loss of membraneintegrity. The events are mediated by "death triggers" which digest anddestroy cell membrane and propagate an injury by producing a cascade ofcell membrane changes. Similar events in tissue culture are vital to thebiologic availability of cells and cell products while still permittingcells to possess the capacity to respond to their environments or eachother. By interfering with the cascade of external and internal eventsinvolving membrane integrity and toxic changes which lead to cell death,injury can be prevented, modified or reversed. This has been a majorrole of anti-inflammatory agents in the past.

The most important presently used clinically effective anti-inflammatorydrugs include the corticosteroids and the non-steroidalanti-inflammatory agents (NSAIAs). These drugs act to controlinflammation and to minimize cell injury by regulating the breakdown ofphospholipids or the action of the products of such breakdown leading tothe formation of prostaglandins and leukotrienes which are produced inincreased quantities in inflammation and promote cell dysfunction andinjury.

In addition, recent studies have demonstrated that cellular and extracellular phospholipases may be activated by the generation of oxygenfree radicals. This can establish a "vicious cycle" as phospholipaseactivation can release free radicals which, in turn, activate morephospholipases. In this regard, free radicals are produced from fattyacids released by the action of phospholipases, which are converted toprostaglandins and leukotrienes. Fatty acids and free radicals are knownto be prime mediators in the cascade of reactions that result inmembrane injury, cell death and inflammation.

In addition to effects involving free radical formation, an additionalrole for phospholipases, particularly phospholipase A₂ (PLA₂), is thatthrough their action promoting fatty acid release, as an example theyproduce arachidonic acid derivatives that promote potassium (K⁺) ionchannel opening which governs the electrophysiologic and secondmessenger responses of the cell. Thus, phospholipase inhibitors canmodulate cell responses to membrane stimulation governing cell function.

One of the hallmarks of inflammation and cell injury is the breakdown ofcellular membrane phospholipid.

Phospholipids are the major structural building blocks of the cellmembrane; they give rise to the barrier-structural and functionalproperties of membranes and their integrity is crucial to normal cellresponsiveness and function.

Phospholipid changes in cell membrane integrity, particularly changes infatty acids at the 2 position, alter the fluidity of cell membranes,their receptor availability and the leakiness or availability ofcellular contents to the external environment. The breakdown ofphospholipid membranes results in "unraveling" or lysis of cells, orresults in holes in the cell membrane, the disruption or enhancement ofion channels, or the loss of membrane bound receptors which destroysintegrity and functional survival.

During inflammation, phospholipases, from whatever source, that arenormally under the control of natural suppressor systems, are activatedto degrade membrane phospholipid which, in turn, generates oxygen freeradicals. A key enzyme which is activated in inflammation isphospholipase A₂ (PLA₂) which acts on phospholipids as enzyme targets torelease free fatty acids. These fatty acids (i.e., arachidonate)released by PLA₂ are converted to potent biologically activemetabolites, prostaglandins and leukotrienes, with the concomitantgeneration of oxygen free radicals. These PLA₂ products have effects onK ion channels and G proteins involved in second messenger cellresponses involved in cell membrane homeostasis. These metabolites,fatty acids and free radicals, are powerful mediators of pathophysiologywhich propagate injury and cell death or permit the nidation, survivaland growth of pathogens or tumor cells.

The role of phospholipases, particularly PLA₂, as membrane targetedenzymes, make them veritable "death triggers" as the expression of theirdegradation activity results in further production of inflammatorymediators leading to further membrane injury which propagates damagewithin the cell itself or into adjacent tissue. Thus, the spread ofinjury from the initial site to contiguous or distant sites can bepromoted by the activation and/or release of PLA₂.

In addition to the intrinsic membrane-related tissue breakdown via theactivation of PLA₂, phospholipases, and particularly PLA₂, are part ofthe normal defensive system of the body. PLA₂ is found in particularlyhigh levels in human white blood cells (WBCs): polymorphonuclearleukocytes (PMNs) and phagocytic cells. WBCs play a role in resistinginfection, but when these cells are mobilized to ward off injury andinfection, PLA₂ is released from adherent and circulating WBCs andproduces local tissue necrosis which increases the extent of initialinjury. In addition, WBCs adhere to blood vessel walls where theyrelease enzymes such as PLA₂. WBCs also generate free radicals and thuspromote damage to the vascular endothelium, lung alveoli or to tissuesites contiguous with WBC infiltration or concentration. Whereinflammation is found, WBCs are usually present in abundance and theWBCs adhere to vascular endothelium, with release and activation of PLA₂resulting in damage to vascular integrity during shock and ischemia.Thus, in spite of being a prime defensive system of the body againstinfection, WBCs can also damage the body by propagating injury andinflammation beyond their normal defensive role.

The classic description of inflammation is "redness and swelling withheat and pain (Celsus, 100 AD)." Inflammation has been defined as thereaction of irritated and damaged tissues which still retain vitality.Inflammation is a process which, at one level, can go on to cell death,tissue necrosis and scarring and at another level, inflammation can beresolved with a return to normalcy and no apparent injury or withminimal changes, i.e., pigmentation, fibrosis or tissue thickening withcollagen formation related to healing and scarring. The process isdynamic, with cell death as one consequence, and recovery, healing andscarring as another. For inflammation to occur as a process, cells mustretain their vitality. Dead or severely compromised cells do not respondto inflammatory reactions. Injury in inflammation can also relate to thelate results of fibrosis and scarring with the loss of blood vessels,tissue elasticity and cosmetic quality.

Inflammation, while a normal process of the body's resistance to injuryand infection, can become aberrant leading-to propagated injury withextensive scarring, tissue death and/or the death of the organism.Within certain limits, the inflammatory reaction is stereotyped and itcannot distinguish between those instances in which the process protectsthe host and those in which the host is harmed.

Microscopically, inflammation is characterized by vasodilation, vascularleakage, enhanced lymphatic flow, platelet vascular adherence andclumping and WBC infiltration and vascular adherence and phagocytosiswith slowing of blood flow, red cell aggregation resulting in theformation of blood clots. Clinically, these local phenomena can beassociated with pain, fever and swelling which can lead to local tissuedestruction (granulation, caseation and necrosis) healing or scarring orto systemic symptoms of pain, fever, shock (prostration) hypotension,leading to death or recovery.

Microscopically, inflammation has been described as related to: (1)atony of the muscle coat of the blood vessel wall; (2) increasedresistance to blood flow related to friction and adhesiveness of bloodelements (i.e., red blood cells, proteins, white blood cells,platelets): and (3) enhanced permeability, i.e., loss of red bloodcells, white blood cells and blood fluid through the vascular wall.

In physiological terms, these are described as hyperemia, edema, bloodstasis, thrombosis, and hemorrhage. Inflammation can be mediated byhumoral substances produced by tissue elements or infectious agents orby changes in pH (acidity) or oxygen concentration. Clinically, pain,fever, malaise, muscle, arterial and visceral spasm as well as headacheand confusion states can accompany inflammation for whatever primarycause.

The above events are often mediated by phospholipase activation,followed by fatty acid release and the formation of free radicals. Theseevents can be endogenous to the matrix of the body, the supporting cellsand tissues that are functionally or systemically integrated or relatedto specialized host defense mediating cells, i.e., induced by whiteblood cells or platelet activity which respond as part of the body'sdefenses and which release phospholipases or free radicals as part oftheir role in resistance to infection, or their place in the normalmaintenance of coagulative or vascular integrity, i.e., the preventionof hemorrhage, thrombosis or ischemia.

Alternatively, phospholipase activity can relate to exogenous enzymeactivity released from infecting pathologic organisms such as viruses,bacteria, Rickettsia, protozoa, or fungi which posses phospholipase asfactors intrinsic to their infectious activity. In this regard,infecting organisms such as bacteria, viruses, Rickettsia, protozoa ortheir toxins can stimulate infected cells or the endogenous defensesystem to release phospholipases, i.e., PLA₂, which can act locally orat distance sites to produce inflammation or tissue damage. In the caseof Naegleria, a pathogenic amoeba with affinity for the brain,destruction of brain membranes induced by phospholipases secreted byNaegleria can occur at sites in the brain distant from where theorganism is localized.

In regard to intrinsic effects of PLA₂, the same is produced inextremely high concentration by inflamed collagenous spinal discs whereits localized action is associated with severe pain and muscle spasm, aspart of low back and cervical cord injury resulting in acute or chronicdiscomfort.

Phospholipases released from infecting organisms or as a result oftissue injury can induce coagulation of blood proteins, producingischemic injury at sites contiguous or distant to the primary disease.In considering the action of phospholipases, it must be recognized thattheir pathologic effects can be both local, regional or systemic. Thisis governed by the phospholipase enzyme released, the level of albumin,natural inhibitors of enzyme action, and factors of diffusion,circulation and tissue vulnerability based on intrinsic inhibitors orthe susceptibility of previously damaged or oxidized membranes orproteins to phospholipase action.

Inflammation is associated with trauma, infection and host defensereactions, i.e., fever, malaise and shock, related to direct bacterialor virus killing or associated immune responses. Immune responses can beboth beneficial, protective or tissue damaging as can be seen in theirbeing responsible for resistance or cure of infection, or on the otherhand, capable of producing autoimmune phenomenon that results inallergy, i.e., asthma, urticaria, host versus graft disease, glomerularnephritis, rheumatic fever, lupus and rheumatoid arthritis.

In regard to the current treatment of inflammation, corticosteroids areeffective anti-inflammatories, but must be used with caution clinicallybecause they are powerful immunosuppressants and inhibitors offibroblast activity necessary for wound and bone repair. In addition,corticosteroids are diabetogenic drugs and their toxic side effectsinvolve interference with wound repair and bone matrix formation, andresult in sodium retention, potassium loss and decreased resistance toinfection. Corticosteroids also have effects on steroid formation, bloodpressure, protein utilization, fat distribution, hair growth and bodyhabitus. Alternatively, the clinically active NSAIAs, such as aspirin,indomethacin, ibuprofen, etc., work by inhibiting the conversion of freefatty acids to prostaglandins. The side effects of NSAIAs includegastric ulceration, kidney dysfunction and Reye's Syndrome, andmetabolites of prostaglandin can be either damaging or protective tocells depending on the structure of the prostaglandin produced orutilized pharmacologically and the route of administration, cell ortissue effected. In addition to effects on inhibition of cyclooxygenase,some of the NSAIAs, including ibuprofen, indomethacin and meclofenamate,directly inhibit PLA₂ activity in vitro.

As discussed previously, in conjunction with fatty acid release, as partof phospholipid cell membrane mediated injury produced by phospholipaseactivation, leukotrienes are generated. These leukotrienes produced frommembrane phospholipid breakdown, damage tissue through direct toxicaction, effects on ionic channels, and associated free radicalformation: or by indirect effects on vascular smooth muscle or vascularendothelial lining via platelet, WBC, endothelial (blood vessel lining)or smooth muscle constricting interactions.

Leukotrienes are responsible for smooth muscle constriction leading tobronchospasm and the asthmatic attacks seen in allergy or infectiousasthma. Thus, there is an ongoing active search for leukotrieneinhibitors for clinical application in the treatment of allergy, asthmaand tissue injury and inflammation.

Because the phospholipase activated biochemical pathway for theformation of prostaglandins and leukotrienes derived from free fattyacids is branched, inhibition of one branch of this pathway, as withNSAIAs, can create an imbalance in these reactive metabolites. Thisimbalance may actually aggravate inflammation and promote cell injury asevidenced by the gastric ulceration side effects of NSAIA'S, which,along with pH changes have intraperitoneal inflammatory effects.

Due to these adverse effects of both steroids and NSAIAs, there iscurrently much clinical medical interest in identifying phospholipaseinhibiting agents that do not have steroidal side effects, but likecorticosteroids modulate the first step leading to the production ofinjurious metabolites, fatty acids and free radicals.

Free radicals, produced by white blood cells, tissue injury or metabolicprocesses, are highly reactive chemical species which, in the case oftissue injury, are most often derived from respiratory oxygen. Oxygen,while necessary for energetics of life, is also a toxin which, as thechemically related superoxide, or as peroxides, can damage tissueinstead of supporting it. Free radicals derived from oxygen are criticalto damage produced by radiation, inflammation, ischemia (loss of bloodsupply) or through excess oxygen inhalation or exposure. As statedpreviously, free radicals are used by white blood cells to destroyinfecting organisms, but can, under circumstances of shock, infectionand ischemia, damage or destroy the tissue they were meant to protect.

Free radicals, induced by radiation, oxygen exposure, chemical agents(i.e., alkylating agents, dioxin, paraquat) or white blood cellreactions may be tissue damaging or important to mutational changesassociated with aging, radiation or chemotherapy injury, the developmentof cancer and hyperimmune proliferative disease such as rheumatoidarthritis. In addition, these reactive chemical species can, throughoxidation of proteins, enhance the vulnerability of proteins to proteasedigestion.

In the prior commonly assigned '941, '330, '739 and '408 applicationsidentified above, it was disclosed that PGBx, a fatty acid polymer, is aspecific PLA₂ inhibitor and prevents the generation of free radicals.PGB_(x) is capable of inhibiting inflammation induced by PLA₂ ; preventsarachidonate release from human PMNs and vascular endothelium andphospholipids; blocks the synthesis of eicosanoid prostaglandinprecursors; protects lipids from auto-oxidation; and decreasesendogenous lipid peroxide formation and oxidation of tissue homogenates.

The PGB_(x) compounds delay aging in houseflies, prolong survival anddecrease the age-related pigment in cardiomyocytes, protect parameciafrom benzpyrene photoactivation lysis, block the production ofsuperoxide from PMN WBCs, protect the myocardium or myocardial cellsfrom anthracycline or high oxygen tension pro-oxidant injury, blockcarrageenin and adjuvant induced inflammation and arthritis, blockplatelet aggregation and blood clotting, and interfere with cytotoxicimmune response; block IL-3 leukokine stimulation of mast cellproliferation in vitro, and spontaneous gamma interferon C2 complementrelease. These compounds can screen out cytotoxic ultraviolet exposureand are systematically and orally active and stable to autoclaving andfiltration sterilization.

The general chemical structure of the PGB_(x) and other oligomersdisclosed in the above-identified '330 application has beenhypothesized; however, the total defined chemistry required forsatisfying regulatory agencies involved in drug development has remainedobscure. Accordingly, what is still needed are pharmacologically activematerials that are free of compounds having isomeric and/or structuralvariations.

SUMMARY OF THE INVENTION

The present invention provides both lipid and/or water soluble,pharmacologically active, structurally defined compounds that are PLA₂inhibitors having antioxidant properties and that are relatively pure orpurifiable. Thus, the invention provides certain relatively purebioactive compounds which are polymers and/or oligomers of fattymoieties that inhibit PLA₂ ; and which bind to the PLA₂ enzyme. Thecompounds of the invention possess at least two cis-double bonds toenhance anti-inflammatory and cytoprotective or tissue protectiveeffects of the compounds by making them at least dually active.

The compounds of the invention may be used for treating orprophylactically inhibiting phospholipase mediated injury and/or injurydue to oxidation. In a specific sense the invention provides agents forpreventing and/or treating inflammation and cell destruction inmammalian tissue and for protection and preservation of biologicmaterial such as food, tissue samples and even wood or cellulose derivedproducts.

The compounds of the invention protect phospholipid cell membranes, aswell as proteins from the effects of oxidative injury or aging. Thesecompounds also inhibit free radical reactions and thereby stabilizeproteins for maintenance of biologic half-life, anti-coagulant activity,and food preservation.

More specifically, the invention provides pharmacologically active,relatively pure or purifiable antioxidant, anti-phospholipid compoundsthat are chemically defined. These compounds are soluble and/ordispersible in a suitable carrier. In accordance with the invention, thecompounds have at least two fatty moieties and no active hydroxyl group.Each fatty moiety has from sixteen to twenty carbon atoms and at leastone cis-unsaturated double bond. In one preferred form, the compounds ofthe invention may have an acid group.

In accordance with the invention the compound may be a derivative ofricinoleic acid having the formula ##STR1## wherein R₁ may be an alkoxygroup which includes an acid group and R₂ may be an alkoxy group whichincludes one of the fatty moieties. More particularly, in the foregoingcompound R₂ may be ##STR2## wherein Y is --O-- or --NH--.

In an alternative form the compound of the invention may be a derivativeof ricinoleic acid having the formula ##STR3## wherein R₁ may be analiphatic group which includes an acid group and one of the fattymoieties and R₂ may be OH or an alkoxy group which includes another oneof the fatty moieties. In this form of the invention, when R₂ is OH, R₁may be ##STR4##

In another alternative form of the invention, the compound may have theformula ##STR5## wherein X is a divalent organic moiety and R₃ may be OHor an alkoxy group which includes one of the fatty moieties. In thisform of the invention R₃ may preferably be ##STR6## wherein Y is --O--or --NH--, and X may preferably be ##STR7##

In yet another alternative form of the invention the compound may be aderivative of ricinoleic acid having the formula ##STR8##

In accordance with the invention, the compound may also have the formula

    R--O--X--O--R

wherein X is a divalent organic moiety which includes an active acidgroup and the R groups may be the same or different, and each may be##STR9##

As yet a further alternative, the compound of the invention may have theformula ##STR10## wherein X is a divalent organic radical which includesan active acid group, the R groups may be the same or different and eachR may be ##STR11## wherein Y is --O-- or --NH--, and X may be ##STR12##and wherein Z is --O--, --S--, --CH₂ -- or --NH--.

In another alternative form the compounds of the invention may have theformula ##STR13## wherein A is an organic or inorganic anionic moiety;R₁ is --CH₂ --O--R, a hydrogen molecule or a C₁ to C₄ aliphatic group;and the R groups may be the same or different and each R is a fattymoiety. In this form of the invention, R may be ##STR14##

Alternatively, the compounds of the invention may have the formula##STR15## wherein the R groups may be the same or different and each Ris a fatty moiety as set forth above. In this form of the invention Zrepresents a C₁ to C₅ aliphatic organic moiety and A represents anorganic acid moiety.

In another alternative form, the compounds of the invention may have thefollowing structural configuration: ##STR16## wherein the R groups maybe the same or different and each is a fatty moiety, and Z is a C₁ to C₅aliphatic organic radical. More specifically, in the foregoing formula,Z may be --CH₂ -- and R may be ##STR17## When the foregoing compoundsare contacted with NaOH, the lactone ring opens to present the followingstructural configuration: ##STR18##

The compounds of the invention may also have the following structuralformula ##STR19## wherein A is a C₁ to C₇ aliphatic moiety, R₁ is--B--R, a C₁ -C₄ alkyl group, a nitro group, an amino group, acarboxylic acid group, a sulfonic acid group, or a hydrogen atom, said Zgroups are the same or different and each is --O-- or --NH--, and each Ris a fatty moiety as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 through 7 illustrate structural configurations and reactionschemes for preparing various embodiments of the novel cytoprotective,pharmacologically antioxidant, anti-phospholipase active compounds ofthe invention;

FIG. 8 is a graph illustrating the ability of ricinoleic acid-sebacylester polymer to inhibit PLA₂ from a variety of sources; and

FIG. 9 is a graph illustrating the ability of the compounds of theinvention to inhibit PLA₂ activity in vitro.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In previous work by Ballou and Cheng (Proc. Natl. Acad. Sci. 80:5203-5207, 1983, IBID 82: 371-375, 1985) and Marki and Franson (Biochem.Biophys. Acta 879:149-156, 1986) it has been reported thatcis-unsaturated, but not saturated fatty acids, inhibit in vitro PLA₂activities derived from human platelets and human polymorphonuclearleucocytes (PMNs). PLA₂ activity has been shown to be inhibited byoleic, linoleic, and arachidonic acids to approximately the same extentindicating that the presence of a single cis-double bond is asinhibitory as multiple cis-double bonds. In contrast, neither fattyacids containing trans-double bonds nor methyl esters of cis-unsaturatedfatty acids are inhibitory of PLA₂ activity. Thus, it has beenhypothesized that the preferred structural characteristics forinhibition of in vitro PLA₂ activity by unesterified fatty acids includeat least one cis-double bond. As was illustrated above, oleic acidinhibits in vitro PLA₂ activity due to the presence of a singlecis-double bond at the C-9 position. However, recent studies havedemonstrated that hydroxylation at the C-12 carbon atom to produce12-hydroxy-oleic acid (ricinoleic acid) eliminates the inhibitoryactivity. Thus, despite the fact that the cis-double bond is intact,this hydroxylation substitution converts an inhibitory fatty acid to anon-inhibitory fatty acid. In fact, ricinoleic acid is a potentinflammatory agent and is used as such in experimental models or as aneffective ingredient of caster oil's cathartic action.

In support of this, a similar change occurs with arachidonic acid, a 20carbon fatty acid with 4 cis-double bonds. This compound is aninhibitory cis-polyunsaturated fatty acid which is converted toprostaglandin B₁ (PGB₁) via the cyclo oxygenase pathway. The productPGB₁ is markedly less inhibitory than the precursor arachidonic acid (byat least 2 orders of magnitude) presumably because it has acquired ahydroxyl group.

Based on the above, it is evident that oxidative reactions canneutralize the PLA₂ inhibitory effect of cis-unsaturated fatty acids. Infact, hydroxylation can make these fatty acids pro-inflammatory. Ofphysiologic and pharmacologic importance to this, data has beenpresented in the '330 application identified above to show thatpro-oxidation of the sarcoplasmic reticulum of muscle predisposes thetissue to phospholipase degradation. The oxidation of phospholipidmembranes enhances the vulnerability of cell membranes to phospholipasedegradation. Phospholipid membranes that have been oxidized atparticular sites may appear intact and maintain functional activity, buttheir oxidation makes them vulnerable to degradation and destruction byPLA₂ or other phospholipases, from endogenous or exogenous sources.

The reversal of PLA₂ inhibition by radical-mediated oxidation ofcis-unsaturated fatty acids may be an important means by which in situPLA₂ s are activated to mediate tissue inflammation, differentiation,and cell death. In support of this, it has been determined thatarachidonic acid binds directly to purified PLA₂ from snake venom.Moreover, when the arachidonic acid is peroxidized by exposure to air at37° C., the arachidonic acid remains bound to the isolated PLA₂ but nolonger inhibits in vitro PLA₂ activity. These results are the focus ofthe discovery of the fact that cellular PLA₂ s are fatty acid bindingproteins, and when the bound fatty acid is cis-unsaturated, the PLA₂will be inhibited. Furthermore, when cis-unsaturated fatty acids areoxidized, PLA₂ enzymic activity is restored, thereby increasing thelevel of "effective" enzyme. The net result when fatty acids areoxidized is an apparent activation of PLA₂ to induce membrane injury,inflammation, cytotoxicity and cell death. This shift from inactive toactive form of PLA₂ signals the onset of the loss of phospholipidmembrane structure.

In related studies, Jung et al. (Biochem. Biophys. Res. Commun.130:559-566, 1985) have shown that the specific hydroxylation of carbon15, but not carbon 5 or 12, of arachidonic acid esterified at the2-position of phosphatidylcholine, increased pancreatic PLA₂ mediatedhydrolysis of the phospholipid by 170%. Thus, oxidative reactions ofcis-unsaturated fatty acids not only "activate" PLA₂ enzyme function byaltering inhibitory fatty acids, but also can increase thesusceptibility of the substrate phospholipid to hydrolysis induced bythe enzyme.

The observations illustrating the enhanced vulnerability of phospholipidmembranes to phospholipase following oxidative and radical mediatedchanges in cell membranes and/or cis-fatty acids have been employed inaccordance with the present invention in the design of novelanti-inflammatory and cytoprotective agents. The invention thus providesa biochemical and synthetic organic approach to controlling theexpression of PLA₂ enzymes and is vital to the maintenance of membranestructure.

It is important to the understanding of the present disclosure torecognize that the number of available methylene interruptedcis-unsaturated double bonds is directly related to the susceptibilityof fatty acids to oxidation. This governs the ability of cis-unsaturatedfatty acids to act as anti-oxidants. This property, in conjunction withthe anti-PLA₂ activity of the fatty moiety compounds of the invention,markedly expands the scope of the anti-inflammatory and cytoprotectiveactivity of the new agents disclosed herein. It is the property of thedual action of these compounds, i.e., their action as PLA₂ inhibitorsand their combined anti-oxidant activity, that provides the spectrum ofanti-inflammatory activity in model systems that have directapplicability to cytoprotection and the control of inflammation andpathophysiology.

In summary, a single cis-double bond in a fatty moiety compound issufficient to inhibit PLA₂ activity in vitro and in situ. The additionof multiple double bonds provides the additional value of an increase inpotent anti-oxidant activity along with PLA₂ inhibitory action. Thepresent invention thus provides compounds characterized by bothanti-PLA₂ and anti-oxidant activity to thereby maximize theanti-inflammatory and cytoprotective action which is the key to theclinical value of the compounds of the invention.

In addition to inhibiting PLA₂ activity, the anti-oxidant action ofthese compounds protects proteins that become increasingly vunerable toattack by proteases due to oxidation. Thus, the cytoprotective PLA₂inhibitors of the invention, which have anti-oxidant activity as well,have value both in stabilizing membrane phospholipid and in inhibitingor preventing protein degradation or denaturation.

In accordance with the invention, the cytoprotective anti-inflammatorycompounds:

(1) bind to the PLA₂ enzyme;

(2) are capable of being continuously hydroxylated to remove theinhibitory action and activate the enzyme without removal of thecomponent from its PLA₂ binding site;

(3) are compounds containing fatty moieties which may have a free acidfunction or at least are preferably ionizable for water solubility andenhanced PLA₂ inhibition;

(4) include cis-double bonds in the fatty moieties to obtain the bestPLA₂ inhibition; and

(5) possess anti-inflammatory activity as a result of inhibiting freeradical activity.

As reported in the cross-referenced patent applications identifiedabove, prior studies have shown that polymers of PGB₁, but not themonomer PGB₁ itself, provide broad protection in a wide range of celland tissue injury models. In contrast to the present disclosure, nobasic mechanism underlying this generalized protective effect wasidentified. But it was clear from these previous studies that polymersor oligomers were more effective than monomers and that a free carboxylfunction in the polymer may be desirable for optimum protectiveactivity.

In recent studies it has been determined that polymers of PGB₁ inhibit abroad range of Ca⁺⁺ -dependent PLA₂ s, including snake, bee, andscorpion venom, and human PLA₂ s derived from synovial fluid, PMNs,platelets, plasma, sperm, endometrium, and herniated discs. At 10 μMconcentrations, PGB₁ (monomer) was not inhibitory while the dimer had1/2 the maximal inhibitory activity. The inhibitory activity of thetrimer is less than that of tetramer while the activity of the latter isequal to that of PGB_(x). Thus, polymerization of prostaglandin B₁, ametabolite of the cis-polyunsaturated fatty acid arachidonate, generatesinhibitors of PLA₂ s.

Similarly, as reported in the cross-referenced applications, polymers ofPGB₁ are equally effective at inhibiting the release of arachidonic acidand platelet activating factor from prelabelled human PMNs andendothelial cells which are reactions critical to the inflammatoryresponse of these cells and which are mediated by endogenous PLA₂activity.

Of particular relevance to the present disclosure, is the fact thatpolymers of PGB₁ also have potent anti-oxidant activity. Arachidonicacid, as noted above, contains 4 cis-unsaturated, methylene interrupted,double bonds which are particularly susceptible to oxidation. Whenarachidonic acid is converted to prostaglandin B₁ by cyclo-oxygenase, anenzyme mediated oxygenation, two of the four original cis-double bondsare retained. These bonds remain in a methylene-interruptedconfiguration, which is the optimal orientation for their oxidation.

The compounds of the present invention block arachidonic acid releasefrom human PMNs and endothelial cells, a reaction mediated by cellularPLA₂ activity. Thus, the compounds of the invention are potent,reversible enzyme-targeted PLA₂ inhibitors, and as such these agentsinhibit the pro-inflammatory response of the human PMN and otherinflammatory cells by inhibition of cellular PLA₂ activity. In addition,the compounds of the invention inhibit free radical activity in cellsand tissue involved in the inflammatory process.

The compounds of the invention also are effective anti-oxidants.Oxidation of phospholipid and arachidonic acid is inhibited in adose-dependent manner. This property not only protects membranes fromradical mediated injury to stabilize function, but also prevents thepreferential hydrolysis of oxidized phospholipid by cellularphospholipases. These results suggest that the compounds of theinvention act to minimize inflammation at its onset and will alsointerrupt the process in progress.

The ability of cis-unsaturated fatty moiety compounds to preventoxidation of proteins will also suppress the expression of cellproteases by maintaining levels of endogenous protease inhibitors. Theseinhibitors, such as α-1-anti-trypsin, are major components of plasma andtheir inhibitory activity is abolished by free radical mediatedoxidation of --SH groups. The anti-oxidant activity of thecis-unsaturated fatty moiety compounds of the present inventionstabilizes α-1-anti-trypsin via the anti-oxidant action. Thus, theanti-oxidant activity of cis-unsaturated fatty moiety compoundsregulates the expression of both lipolytic and proteolytic enzymes(i.e., elastase) by preventing the oxidation of both substrate andendogenous inhibitors.

The 12-hydroxylation of oleic acid yields ricinoleic acid. Thus, aninhibitory fatty acid is converted into a non-inhibitory hydroxylatedderivative. However, the --OH moiety can be used to generate inhibitorycompounds. The hydroxylated derivatives thus provide building blockprecursors for the synthesis of active cytoprotective agents with PLA₂inhibiting activity. Indeed, when ricinoleate is polymerized by heat insulfuric acid, the crude unfractionated product inhibits in vitro and insitu PLA₂ activity.

In effect, the inactive non-inhibiting monomers are converted toextremely active PLA₂ inhibitors. Moreover, active pro-inflammatorymediators are converted structurally to provide anti-inflammatorycompounds.

PLA₂ inhibitors such as derivatives of ricinoleic acid that do not havesignificant anti-oxidant activity have limited anti-inflammatory effect.The presence of the unsaturated bonds of the cis-unsaturated fattymoiety compounds enhances the anti-inflammatory activity. This indicatesthat inflammatory reactions may vary in their characteristics whereinsome inflammatory response can be inhibited or attenuated by PLA₂inhibition alone, while other reactions require anti-oxidant activityalong with PLA₂ inhibition. Thus, in accordance with the invention, toobtain broad spectrum anti-inflammatory action, the dual action ofanti-PLA₂ and anti-oxidant activity is preferred. Polyunsaturatedcompounds constructed from unsaturated fatty acids and other unsaturatedfatty compounds provide potent anti-oxidant activity as well asanti-PLA₂ action.

In a general sense, the invention provides pharmacologically active,antiphospholipase compounds that are chemically defined and purifiable.Preferably the compounds of the invention are water solubleantioxidants. From a functional viewpoint, the preferred compounds haveat least two fatty moieties and no active hydroxy group. Each fattymoiety should preferably have sixteen to twenty carbon atoms and atleast one cis-unsaturated double bond. The compounds may also have atleast one active acid group. In one preferred form, the compound may bea derivative of ricinoleic acid having a structural configuration as setforth in FIG. 1 of the drawings where R₁ is an alkoxy group whichincludes an active acid group and R₁ is an alkoxy group which includesone of the fatty moieties. In one case the R₁ moiety may be obtained byesterification of the 12-position hydroxy group of ricinoleic acid withan acid group of a divalent acid such as sebacic acid, fumaric acid,maleic acid, oxalic acid or succinic acid, and the R₂ moiety may beobtained by esterification of the 1-position carboxy group of ricinoleicacid with the hydroxyl group of another ricinoleic acid molecule or ofsome other cis-unsaturated fatty alcohol such as, for example, oleylalcohol, linoleyl alcohol, linolenyl alcohol, arachidonyl alcohol orcis-5, 8, 11, 14, 17-eicosapentaenoyl alcohol. Alternatively, the R₂moiety may be obtained by amidification of the 1-position carboxy groupof ricinoleic acid with the amine group of a cis-unsaturated fatty aminesuch as oleyl amine, linoleyl amine, linolenyl amine, arachidonyl amineor cis-5, 8, 11, 14, 17-eicosapentaenoyl amine. Thus, for example, R₂may be a moiety having one of the following configurations: ##STR20##wherein Y may be --O-- or --NH--.

Alternatively, the compound may have the generic structural formula asset forth in FIG. 1; however, in this; case R₁ may be an aliphatic groupwhich includes an active acid group and one of the cis-unsaturated fattymoieties and R₂ may be either a hydroxy group or an alkoxy group whichincludes fatty moieties. In this alternative case, when R₂ is a hydroxygroup R₁ may be derived by esterification of the 12-position hydroxygroup of the ricinoleic acid, with, for example, the acid group of oleicacid, linoleic acid, linolenic acid, arachidonic acid or cis-5, 8, 11,14, 17-eicosapentaenoic acid. Thus, in this case, R₁ may be a fattymoiety having one of the following configurations: ##STR21##

With reference to the structural configuration presented in FIG. 1, inanother alternative embodiment R₁ may be ##STR22## wherein X is adivalent organic moiety and R₃ is OH or an alkoxy group that includes acis-unsaturated fatty moiety. When R₃ is OH, either X or R₁ must includea cis-unsaturated fatty moiety. On the other hand, in accordance with analternative embodiment of the invention, R₃ may be a moiety having oneof the following configurations: ##STR23## wherein Y is --O-- or --NH--.

In the foregoing embodiment, when R₁ of the structure of FIG. 1 is##STR24## X, for example, may be one of the following divalent moieties:##STR25##

When R₂ of the FIG. 1 structure is derived by esterifying the12-position OH of ricinoleic acid with the 1-position carboxy group ofanother molecule of ricinoleic acid, the resultant structure will be asshown in FIG. 3. In this case the OH at the 12-position of the secondricinoleic acid molecule must be rendered non-inhibitory. This may bedone by esterification to cause R to be, for example, one of thefollowing moieties: ##STR26##

In accordance with another embodiment of the invention, the structuralconfiguration of the compound may be

    R.sub.1 --O--X--O--R.sub.1

wherein X is a divalent organic moiety which may include an active acidgroup and the R₁ groups may be the same or different and each may, forexample, be one of the following moieties: ##STR27##

In this form of the invention, X may be derived by esterification of thehydroxy group of tartaric acid whereby X will have the followingstructural configuration: ##STR28## An appropriate reaction scheme forthis procedure is illustrated in FIG. 5. Each ##STR29## moiety in theFIG. 5 compound is represented by an R₁ group in the compound

    R.sub.1 --O--X--O--R.sub.1.

In yet another alternative form, the compounds of the invention may havethe structure ##STR30## wherein X is a divalent organic moiety which mayinclude an active acid group and the R₁ groups, which may be the same ordifferent, may, for example, be one of the following moieties: ##STR31##wherein Y is --O-- or --NH--.

In this embodiment, X may be ##STR32## wherein Z may be --O--, --S--,--CH₂ -- or --NH--. The compounds of this embodiment may be preparedusing a reaction scheme as illustrated in FIG. 4 where the producedcompound is shown as having carboxyl groups at the 2 and 3 positions ofthe ring. However, as will be appreciated by those skilled in the art,the reaction scheme of FIG. 4 may be conducted so as to provide any oneof several isomeric compounds, i.e., with the carboxyl groups at the 2and 3 positions as shown or alternatively with the carboxyl groups atthe 1 and 2 positions, at the 1 and 3 positions or at the 1 and 4positions. Each R--Y-- moiety in the FIG. 4 compound is represented byan R₁ group in the compound ##STR33##

The invention contemplates a variety of configurations including, forexample, dimers, trimers and tetramers. Dimers are illustrated in FIGS.1, 2, 4 and 5, trimers are illustrated in FIGS. 1 and 3 and tetramersare illustrated in FIGS. 6 and 7. The structures illustrated in FIGS. 1and 3 and the reaction schemes of FIGS. 4 and 5 have been discussedabove. The structure illustrated in FIG. 2 is a dimer of ricinoleic acidprepared by esterifying the 12-position hydroxy groups of two moleculesof ricinoleic acid with the carboxy groups of a diacid such as sebacicacid, fumaric acid, maleic acid, oxalic acid or succinic acid, forexample.

With reference to FIG. 6, the compounds produced in accordance with FIG.5 may be linked together by esterification through one of the free acidgroups. Thus, the acid groups may be converted to acid chloride groupsand reacted with hydroxy or amine groups of a divalent compound havingthe form

    H--Y--R.sub.1 --Y--H,

wherein Y is --O-- or --NH-- and R₁ is a divalent preferably aliphatic,organic moiety. As before, the R groups of the compound formed asillustrated in FIG. 6 may be the same or different and may preferably becis-unsaturated fatty moieties.

The reaction scheme illustrated in FIG. 7 may employ any one of theisomeric compounds that may be formed in accordance with the reactionscheme of FIG. 4 as discussed above. Thus, the free acid groups of oneof the FIG. 4 isomeric compounds are converted to the acid chloride oranhydride and esterified with the hydroxy or amino groups of a bivalentorganic moiety.

In each case, the compounds of the invention include at least twocis-unsaturated C₁₆ -C₂₀ straight chain fatty moieties and have noactive hydroxy group. Desirably, each compound may also include at leastone active acid group.

The novel water soluble and/or lipid soluble pharmacologically active,pure or purifiable antioxidant, antiphospholipase compounds provided inaccordance with principles and concepts of the invention may be preparedas outlined in the following specific examples.

EXAMPLE I Diricinoleic Acid Fumarate Diester (RAFARA)

In a 500 ml single-necked round-bottom flask, 5.0 g of crude ricinoleicacid (Tokyo Kasei Co., 80% content) is dissolved in 180 ml of CH₂ Cl₂containing 2.8 g of NaHCO₃. 6.5 ml of fumaryl chloride (42 mmoles) isadded dropwise over 2 minutes under vigorous stirring by a magneticstirrer at room temperature. The mixture is stirred for 88 hours andthen 100 ml of 5% aqueous NaHCO₃ is added slowly to the reaction flaskwhile cooling with an ice bath. The mixture is poured into a 1000 mlbeaker containing 300 ml of 2% NaHCO₃. Upon standing overnight, the CH₂Cl₂ layer is separated and acidified with 12N HCl until the pH is 1. TheCH₂ Cl₂ solution is washed three times with distilled H₂ O, dried overanhydrous MgSO₄, and the CH₂ Cl₂ is removed by rotoevaporation at 35° C.A yellow oily material is obtained.

This crude product is purified by column chromatography (Aldrich 230-400mesh 1×70 cm) with petroleum ether-ethyl acetate (6:4) as eluent. Underset conditions, dimer (I) is collected with one spot shown on silica gelTLC plate, R_(f) 0.26, (Baker-flex Silica Gel LB-F, 6:4 petroleum ether(bp 50-110° C.)-ethyl acetate).

The product, which may be called RAFARA, has the following structuralconfiguration: ##STR34##

EXAMPLE II Ricinoleic acid-Linoleic Acid Ester (RALA)

Ricinoleic acid 1.0 q (3.4 mmoles, Sigma) is dissolved in 10 ml ofpropylene oxide and placed in a 100 ml round-bottomed, single-neckedflask equipped with a Teflon-coated magnetic stirring bar. The solutionis cooled to 0° C. on an ice-water bath, and 2.0 g linoleoyl chloride(6.8 moles) in 5 ml of anhydrous methylene chloride is added dropwise.The mixture is warmed to room temperature and stirred for 3 days. Afterremoving the solvent by rotoevaporation, the residue is dissolved in 150ml of methylene chloride. The mixture is washed with 200 ml of 5%aqueous NaHCO₃ three times, and then the methylene chloride layer isseparated and dried over anhydrous magnesium sulfate. After removal ofthe methylene chloride, the crude product is purified by columnchromatography using silica gel (Aldrich, 230-400 mesh, 1.0×70 cm).Elution is performed using 8:2 chloroform-ethyl acetate as thedeveloping solvent. The product (RALA) obtained shows one spot on TLC,R_(f) 0.54 (Baker-flex Silica Gel lB-F, 8:2 chloroform-ethyl acetate)and has the following structural configuration: ##STR35##

EXAMPLE III Ricinoleic Acid-Oleic Acid Ester (RAOA)

0.5 g ricinoleic acid (1.7 mmoles, Sigma) is dissolved in 6 ml ofanhydrous CH₂ Cl₂ and placed in a 100 ml round-bottomed, single-neckedflask equipped with a Teflon-coated magnetic stirring bar. Afteraddition of 0.15 g pyridine (1.7 mmoles), the solution is cooled to 0°C. on an ice-water bath, and 0.5 g oleoyl chloride (1.7 mmoles, Sigma)in 4 ml of anhydrous CH₂ Cl₂ is added dropwise. The mixture is warmed toroom temperature and stirred for 2 days. The solvent is removed byrotoevaporation and 500 ml of anhydrous ether is added to the residue.The white precipitate formed is removed by filtration. Ether is removedby rotoevaporation and 50 ml of anhydrous CH₂ Cl₂ is added to the oilyresidue. The CH₂ Cl₂ layer is washed three times with 50 ml portions of5% aqueous NaHCO₃. The CH₂ Cl₂ layer is dried and the solvent removed byrotoevaporation. The crude product is purified by column chromatographyusing silica gel (Aldrich, 230-400 mesh, 1.0×55 cm). Elution isperformed using 8:2 petroleum ether (bp 50-100° C.)-ethyl acetate as thedeveloping solvent. The fractions containing the product are combinedand the solvent is evaporated. The product shows only one spot on TLC,R_(f) 0.51 (Baker-flex Silica Gel lB-F, 8:2:1 petroleum ether-ethylacetate-acetic acid).

The product RAOA has the following structural configuration: ##STR36##

EXAMPLE IV Oleyl Alcohol-Ricinoleate-Fumaric Acid Diester (OAlRAFA)

2.2 g of crude ricinoleic acid-oleyl ester, (4 mmoles, based on theassumption that the material is pure, Tokyo Kasei Co.), is dissolved in10 ml of anhydrous CH₂ Cl₂ and 10 ml of propylene oxide, and placed in a100 ml round-bottomed three-necked flask equipped with a drying tube anda Teflon-coated magnetic stirring bar. The solution is cooled to 0° C.,and 3.04 g fumaryl chloride (20 mmoles) in 10 ml of anhydrous CH₂ Cl₂ isadded dropwise from an additional funnel over 10 minutes. The reactionmixture is warmed to room temperature and stirred for three days. Afterremoval of solvent by rotoevaporation, the residue is dissolved in 100ml of ethyl acetate. The ethyl acetate layer is washed 5 times with 75ml portions of 5% aqueous NaHCO₃. The ethyl acetate layer is separatedand dried over anhydrous sodium sulfate. Ethyl acetate is removed byrotoevaporation to yield a brownish oily residue. This crude product ispurified by column chromatography using silica gel (Aldrich, 230-400mesh, 1.0×55 cm). Elution is performed using 8:2 petroleum ether (bp50-110° C.)-ethyl acetate as developing solvent. The product shows onlyone spot on TLC, R_(f) 0.39 (Baker-flex Silica Gel ;B-F, 8:2 petroleumether-ethyl acetate).

The product OAlRAFA has the following structural configuration:##STR37##

EXAMPLE V Ricinoleic Acid-Sebacyl Ester (RISE-1)

5 g of ricinoleic acid (16.75 mmole, Sigma) is added to a mixture of 200ml of methylene chloride and 2.8 g (33.5 mmole) of sodium bicarbonatecontained in a 500 mL two-necked, round-bottom flask equipped with anaddition funnel and drying tube. This mixture is stirred by means of amagnetic stir bar while 14.27 ml (67 mmole) of sebacoyl chloride areadded dropwise over a 10 minute period. The mixture is continuouslystirred for three days at room temperature. The mixture is then washed 5times with 500 ml of saturated sodium chloride and is then treatedcarefully with 500 ml of saturated sodium bicarbonate solution. Theorganic solvent layer is separated and the remaining aqueous layer iswashed two times with 100 ml portions of ethyl ether. The pH of theaqueous solution is then adjusted to 1.5 with 6N hydrochloric acid at 0°C. and the mixture is extracted with 200 ml of ethyl acetate. The ethylacetate extract is dried over anhydrous sodium sulfate, filtered, andthe solvent removed with a rotor evaporator. The residue is dissolved in100 ml of a warm toluene/tetrahydrofuran (3:1 v/v) mixture. Aftercooling overnight a white precipitate of sebacic acid ester ofricinoleic acid is obtained and removed by filtration. The product ischromatographed on a silica gel column (Aldrich, 230-400 mesh, 1.0×70cm) with toluene/tetrahydrofuran (3:1 v/v) mixture as eluent. The elutedproduct is recovered by is rotoevaporation of the solvent.Chromatography of this product is performed on a silica gel column(Aldrich, 230-400 mesh, 1.0×70 cm), using a petroleum either (bp 50-110°C.)/ethyl acetate/acetic acid (80/30/1 v/v/v) mixture as eluent. Theproduct which shows one spot on silica gel TLC plate, R_(f) =0.33(Baker-flex Silica Gel LB-F, petroleum ether (bp 50-110° C.)/ethylacetate/acetic acid (80/30/1 mixture)), is collected as a clear waxymaterial (mp 70-72° C.).

The product, which may be called RISE-1, has the following structuralconfiguration: ##STR38##

RISE-1 is sebacic acid ester-linked to ricinoleic acid via the 12-OH.RISE-1 inhibits PLA₂ activity in vitro and in situ, and inhibits PLA₂induced mouse paw edema. But like pure ricinoleic acid compounds, RISE-1has no antioxidant activity and does not inhibit rat adjuvant inducedarthritis. Limited antioxidant activity is predicted in this structuredue to the presence of only a single double bond.

The selective activity of RISE-1 provides a dose related, selective invitro inhibition of PLA₂ derived from plasma, sperm, platelets, humansynovial fluid and N. naja cobra venom phospholipase. This isillustrated in FIG. 8 of the drawings. Importantly, the wide range ofPLA₂ enzymes inhibited, i.e., from different sources: human synovialfluid, human plasma, platelets, and sperm suggests that RISE-1 andsimilar compounds are useful both specifically and non-specifically asinhibitors of this enzyme regardless of enzyme source.

Table 1 set forth below shows the in vivo inhibition by RISE-1 ofinflammation induced by the intra-articular injection of human synovialfluid PLA₂ (SF-PLA₂) into the mouse paw. The mouse paw edema isinhibited in dose related fashion by increasing concentrations of RISE-1given orally 1.0 hours before induction of the edema by SF-PLA₂.

                  TABLE 1                                                         ______________________________________                                        Inhibition of Human SF-PLA.sub.2 Induced Mouse Paw Edema by Oral                Administration of RISE-1                                                                    % Increase Weight                                                                           % Protection                                    ______________________________________                                        PLA.sub.2 (Control) 61%            0%                                           PLA.sub.2 + RISE-1 10 mg/kg 47% 23%                                           PLA.sub.2 + RISE-1 30 mg/kg 38% 38%                                           PLA.sub.2 + RISE-1 100 mg/kg  31% 49%                                       ______________________________________                                         Conclusion: RISE1 has oral antiinflammatory activity in this model of         edema.                                                                        Tables 2 and 3 set forth below show the absence of protection provided by     RISE1 against spontaneous oxidation of phosphatidylethanolamine (Table 2)     and against arachidonic acid hydroperoxide formation (Table 3).          

                  TABLE 2                                                         ______________________________________                                        Lack of Effect of RISE-1 on the Autooxidation of                                Phosphatidylethanolamine                                                                     Time                                                           Sample (Hrs) % Unoxidized PE % Oxidized PE                                  ______________________________________                                        PE (control)      0      95.8%       1.4%                                       PE  24 51.1% 39.3%                                                            PE + RISE-1  25 uM 24 50.6% 42.0%                                             PE + RISE-1 100 uM 24 49.6% 42.4%                                           ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Oxidation of Arachidonic Acid (as Measured by Decreased                         Turbidity and Hydroperoxide Formation)                                                      Turbidity    Hydroperoxide                                      (430 nm) (nmols)                                                                          Time (hrs)                                                                      0       24       0     24                                     ______________________________________                                        Control             1.38    .387   100   378                                    Control + RISE-1 25 uM 1.33 .524 109 437                                      Control + RISE-1 50 uM 1.47 .348 109 428                                      Control + BHT 100 uM  1.31 1.283  119 112                                   ______________________________________                                         BHT = betahydroxytoluene                                                 

From the data presented in Tables 2 and 3 it can be concluded thatRISE-1 has no effect on the autooxidation of arachidonic acid orphosphatidylethanolamine. This contrasts vividly with the effect ofother fatty moiety polymers of the invention in this regard and theproducts of Examples II (RALA), III (RAOA) and IV (OAlRAFA) are shown inTable 4 below to be effective inhibitors for the auto-oxidation ofphosphatidylethanolamine.

                  TABLE 4                                                         ______________________________________                                        Effect of fatty moiety compounds on the auto-oxidation of                       phosphatidylethanolamine                                                                     Percent Protection                                           Concentration (μM)                                                                      RAOA       RALA    OAlRAFA                                       ______________________________________                                         25          16%        13%     28%                                              50 16% 22% 24%                                                               100 13% 38% 31%                                                               250 22% 53% 47%                                                               500 27% 73% 62%                                                             ______________________________________                                    

On the other hand, as shown in FIG. 9 of the drawings, RISE-1, RAOA,RALA and OAlRAFA all effectively inhibit human synovial PLA₂ activity ina dose-dependent manner where each compound, prepared as a sodium salt,has an IC₅₀ of approximately 5 to 10 μM.

The data set forth below in Table 5 illustrates the fact that RISE-1, ina dose related fashion, inhibits arachidonic release from prelabelledhuman polymorphonuclear leukocytes (PMNs), indicating that RISE-1,despite absence of antioxidant activity, is capable of modulation, i.e.,blocking, in dose-related fashion, of the release of arachidonic acidfrom stimulated white blood cells (PMNs).

                  TABLE 5                                                         ______________________________________                                        Inhibition of Arachidonic Acid Release from Prelabelled                         Human PMNs                                                                                       % Inhibition                                             ______________________________________                                        PMNs alone                0%                                                    PMNs + RISE-1 25 uM  0%                                                       PMNs + RISE-1 50 uM 19%                                                       PMNs + RISE-1 75 uM 38%                                                       PMNs + RISE-1 100 uM  72%                                                   ______________________________________                                    

Sebacic acid esterified to ricinoleate (RISE-1) lacks antioxidantactivity, but inhibits PLA₂ activity and is anti-inflammatory in thePLA₂ -induced mouse paw edema model. The lack of anti-oxidant activityis believed to be due to the absence of a cis-double bond in the sebacicacid moiety. But when the esterified moiety is either oleic acid (18carbons, 1 cis-double bond) or linoleic acid (18 carbons, 2 cis-doublebonds) the products now have anti-oxidant activity. And the anti-oxidantactivity tends to increase with the number of double bonds (see Table 4and FIG. 9).

In addition to the foregoing compounds, many of which comprisederivatives of ricinoleic acids, additional compounds are includedwithin the broad scope of the invention. Some of these compounds aredefined structurally by the following generic formula: ##STR39## wherein⁻ A is an organic or inorganic anionic moiety; wherein R₁ is --CH₂--O--R, a hydrogen molecule or a C₁ to C₄ aliphatic group; and whereinthe R groups may be the same or different and each R group is a fattymoiety.

In this form of the invention the R groups may have one of the followingstructural formulations: ##STR40##

A method for preparing a particularly preferred compound having theforegoing structural configuration is described in Example VI.

EXAMPLE VI P-Toluene Sulfonate Salt of Tris Trioleate

0.54 g (4.4 mmoles) of tris(hydroxymethyl)aminomethane (Tris, Aldrich),5.0 g (17.7 moles) of oleic acid (Aldrich), and 1.26 g (6.6 mmoles) ofp-toluenesulfonic acid monohydrate (Sigma) are mixed in 50 ml of tolueneand placed in a 100 ml round-bottomed single-necked flask equipped witha Dean-Stark trap and a Teflon-coated magnetic stirring bar. Afterbubbling the reaction mixture with N₂ gas for 10 minutes, the reactionmixture is heated to reflux. The reaction is continued until astoichiometric amount of water is recovered (0.38 ml). After removal ofa small amount of undissolved material by filtration, the toluene isremoved by rotoevaporation to yield a white waxy product. This productis purified on a silica gel column (Aldrich 230-400 mesh, 2.0×55 cm)with 8:2 petroleum ether (bp 60-90° C.)-ethyl acetate as developingsolvent. After eluting with developing solvent the top uncolored layeris carefully removed and the product is extracted with ethyl acetatefrom the silica gel. The solvent is removed by rotoevaporation and theproduct is recovered as a p-toluene sulfonic acid salt of tris trioleatehaving the following structural configuration: ##STR41##

In this procedure, the amine group of the Tris is protected fromreacting with the fatty acid because it is in the form of a p-toluenesulfonate salt. Moreover, the p-toluene sulfonic acid acts as a catalystfor the esterification between the alcohol functions on the Tris and thefatty acid.

Another class of compounds within the generic scope of the invention hasthe following general structural formulation: ##STR42## wherein the Rgroups may be the same or different and each R may be a fatty moiety,wherein Z may be a divalent C₁ to C₅ aliphatic organic radical, andwherein A may be an organic acid moiety. In this case also the R groupsmay have structural formulations as set forth in the precedingparagraph. Preferably Z may be --(CH₂)_(n) -- (wherein n is 1 to 5),##STR43## and A may be a carboxyl group or a sulfonyl group. Methods forpreparing compounds within this group of compounds are set forth inExamples VII and VIII below.

EXAMPLE VII TES Trioleate

To a 250 ml single-neck, round-bottom flask is added 1.0 g (4.36 mmoles)of 2-[tris(hydroxylmethyl)-methylamino]-1-ethanesulfonic acid (TES;Aldrich; 99% purity) and 25.0 ml of anhydrous dimethyl formamide (DMF).The flask contents are then cooled to 0° C. in an ice-water bath. 5.25 g(17.45 mmoles) of oleoyl chloride (Aldrich, technical grade) is addeddropwise over a 5 minute period. The reaction mixture is stirred at roomtemperature from 4 days. The DMF is removed by distillation at 40-45° C.under reduced pressure. The residue is a viscous oil which istransferred to a flask containing 200 ml acetone and vigorously stirreduntil a slightly cloudy solution is formed. This solution isrefrigerated overnight. The precipitate formed is collected byfiltration and washed with distilled acetone (20 ml) five times. Theproduct is dried in vacuo at room temperature for 24 h (2.32 g, 52%) andhas the following structural configuration: ##STR44##

EXAMPLE VIII Tris Trioleate Maleic Acid Amide

2.0 g (1.96 mmoles) of tris trioleate is dissolved in 10 ml of anhydrousCH₂ Cl₂ in a 50 ml single-neck round-bottom flask. The solution iscooled to 0° C. in an ice-water bath and 0.42 g (1.96 mmoles) of CH₃ ONa(Aldrich, 25% by weight in CH₃ OH) is added dropwise under vigorousstirring. After 30 minutes the solvents are removed by rotoevaporationand the residue dried in vacuo at room temperature for 24 hours. To thismaterial, 10 ml of freshly distilled CH₂ Cl₂ and 0.37 g (3.90 mmoles) ofrecrystallized maleic anhydride is added. The reaction mixture isstirred at room temperature for 24 hours. The methylene chloride isremoved by rotoevaporation and the viscous oil obtained is transferredto a 250 ml beaker containing 100 ml acetone and 5 ml of H₂ O. This isvigorously stirred until a clear solution forms. The solution isrefrigerated overnight and until a viscous material forms at the bottomof the beaker. The upper layer is recovered and the acetonerotoevaporated. The residue is stirred with 10 ml CHCl₃ for 15 minutesand the insoluble material is removed by filtration. The CHCl₃ isremoved by rotoevaporation and the residual oil is then similarlytreated with benzene to further remove impurities. The final product isa waxy-like material (0.82 gm 37%) having the following structuralconfiguration: ##STR45##

Yet another group of compounds within the generic scope of the inventionmay be described by the following generic structural configuration##STR46## wherein the R groups may be the same or different and each isa fatty moiety and Z is a C₁ to C₅ aliphatic organic radical. In thisform of the invention the R groups may be ##STR47##

A method for preparing a compound within this group of compounds is setforth in Example IX below.

EXAMPLE IX Tricinyl Trioleate

0.67 g (3.75 mmoles) of N-[tris(hydroxymethyl)methyl]glycine (Tricine;Aldrich) is suspended in 15 ml of anhydrous DMF and placed in a 100 mlround-bottom single-necked flask equipped with a Teflon-coated magneticstirring bar. The suspension is cooled to 0° C. in an ice-water bath,and 5.6 g (18.75 mmoles) of oleoyl chloride is added dropwise. Asolution containing 15 ml of anhydrous CH₂ C₂ and 2.7 g (22.5 mmoles) of4-dimethylaminopyridine is added to the reaction mixture and theresultant admixture is warmed to room temperature, and stirred for 22hours. The admixture is filtered and the solvents are removed underreduced pressure. 200 ml of ethyl acetate is added to the residue andinsoluble material is removed by filtration. The filtrate is washed with100 ml portions of 5% NaHCO₃ aqueous solution (saturated with NaCl)three times. The ethyl acetate layer is dried over magnesium sulfate,and then the ethyl acetate is rotoevaporation to yield a crude productwhich is then purified by chromatography using a silica gel column(Aldrich, 230-400 mesh, 2.0×55 cm) and 8:2 petroleum ether (bp 60-90°C.)-ethyl acetate as developing solvent. The fractions containing theproduct are combined and the solvent is evaporated. The product, whichmay be referred to as an amide-diester lactone, shows only one spot onTLC, R_(f) 0.44 (Aldrich pre-coated silica gel LC sheet: 8:2 petroleumether-ethyl acetate) and has the following structural configuration:##STR48##

If the foregoing product is contacted with NaOH during samplepreparation, the ring opens to present an amide diester hydroxy acidcompound having the following structural configuration: ##STR49##

Still another group of compounds which embody the concepts andprinciples of the invention may be described by the general formula:##STR50## wherein A is a C₁ -C₇ aliphatic group, R₁ may be --Z--R, a C₁-C₄ alkyl group, a nitro group, an amino group, a carboxylic acid group,a sulfonic acid group, or a hydrogen atom, the Z groups, which may bethe same or different, may be --O-- or --NH--, and the R groups, whichalso may be the same or different, may be fatty moieties as describedabove.

Other compounds in the foregoing class of compounds may be representedby the following subgeneric structural formulas: ##STR51##

As can be appreciated, the compounds in this class may be prepared frommultivalent hydroxides and amides by esterification and/or amidificationusing an appropriate fatty acid compound. Suitable starting materialsinclude 2-amino-2-hydroxymethyl-1-hydroxybutane;1,3-diamino-2-hydroxypropane; 1,3dihydroxy-2-amino-2-hydroxymethylpropane; and 1,3dihydroxy-2,2-dihydroxymethylpropane. One shortcoming of the compoundsof this class is their relative lack of solubility in water. However,suitable dose forms may be prepared utilizing DMSO as a solvent.

A procedure for preparing a specific compound in this class is set forthin Example X.

EXAMPLE X 1,3-Diamino-2-hydroxypropane Oleoyl Diamide ester

In a 1 liter reaction flask, 20 g (0.22 mole) of 1,3diamino-2-hydroxypropane are dissolved in 500 ml of CH₂ Cl₂ containing88.1 g (0.72 mole) dimethylaminopyridine. The solution is cooled in anice bath while 213.6 g (0.71 mole) of oleoyl chloride (70%, Aldrich) isadded with stirring over a 2 hour period. The reaction mixture is thenstirred at room temperature for 70 hour. The precipitated salts areremoved by filtration and the solvent is removed by rotoevaporation. Theresidual oil is dissolved in 600 ml of ethyl acetate and the insolublematerial is removed by filtration. The ethyl acetate is washed with asaturated NaCl/H₂ O solution (3 times). The non-aqueous solution isdried over anhydrous MgSO₄, filtered, and the solvent removed byrotoevaporation. The oil obtained is washed with 250 ml of methanol (4times). The residue is taken up in 800 ml of ethyl acetate treated with15 g of charcoal and filtered. The solution is passed through a silicagel column (3.0×10 cm) and the recovered solution is concentrated toyield a viscous light yellow oil (141.6 g). TLC gave one spot and thestructure is verified by IR, H and ¹³ C NMR. The structuralconfiguration is as follows: ##STR52##

The relative abilities of some of the compounds described above toperform as anti-oxidants and as anti-PLA₂ agents is illustrated below inTABLE 6 wherein the polymers compared are as follows:

    ______________________________________                                        PX-1          (PGB.sub.x)                                                       PX-2 (ricinoleate/fumarate monomer)                                           PX-3 (EXAMPLE III dimer)                                                      PX-4 (EXAMPLE II dimer)                                                       PX-5 (EXAMPLE IV dimer)                                                       PX-6 (ricinoleate/ricinoleate dimer)                                          PX-7 (EXAMPLE I dimer)                                                        PX-8 (ricinoleate/oleate/maleate dimer)                                       PX-9 (tartrate/linoleate/linoleate dimer)                                     PX-10 (tartrate/oleate/oleate dimer)                                          PX-11 (EXAMPLE IX trimer)                                                     PX-12 (EXAMPLE VI trimer)                                                     PX-13 (EXAMPLE VII trimer)                                                    PX-14 (tris trilinoleate trimer)                                              PX-15 (tricine trilinoleate trimer)                                         ______________________________________                                    

The terms monomer, dimer and trimer in the foregoing list and as usedthroughout the present description define the number of C₁₆ to C₂₀ fattymoieties present in the particular molecule. That is to say, a monomerhas one such fatty moiety, a dimer has two, a trimer has three, etc.

                  TABLE 6                                                         ______________________________________                                        Comparative Table: Fatty Moiety Polymers (PX)                                                   IC-50          IC-50                                          Compound Anti-Oxidant  Anti-PLA.sub.2                                         Compound Activity  Activity                                                 ______________________________________                                        PX-1          40 uM            1-5  uM                                          PX-2 >500 uM   nd*                                                            PX-3 >500 uM  7.2 uM                                                          PX-4 210 uM  10.0 uM                                                          Px-5 300 uM  5.0 uM                                                           PX-6 300 uM  4.5 uM                                                           PX-7 130 uM  7.0 uM                                                           PX-8 140 uM  3.5 uM                                                           PX-9 100 uM  7.5 uM                                                           PX-10 60 uM  7.0 uM                                                           PX-11 145 uM  2.6 uM                                                          PX-12 50 uM  1.5 uM                                                           PX-13 60 uM  2.2 uM **                                                        PX-14 70 uM  4.4 uM                                                           PX-15 nd *  19.2 uM                                                         ______________________________________                                         * nd = not detectable at ≦ 500 uM                                      ** most effective vs PLA.sub.2induced mouse paw edema; administered singl     dose orally: IC50 approx 15 mg/kg                                        

The polymers (dimers, trimers, tetramers, etc.) of cis-unsaturated fattyacids and the other compounds having at least two cis-unsaturatedstraight chain fatty radicals, as described above, affect fundamentalmembrane phospholipid reactions of phospholipase-induced degradation andfree radical peroxidation. The discovery of these dual properties,anti-phospholipase and anti-oxidant activities of these compoundsestablishes a sound scientific basis for the molecular action thereof inprotecting the cell and its membrane from injury. The data set forthabove confirms, with experimental results, that these compounds arepotent anti-inflammatory and cytoprotective agents.

The appropriate dosage of the cis-unsaturated fatty moiety compounds ofthe invention for treatment of mammals, including humans, againstphospholipase mediated injury and/or inflammation should be in the rangeof from about 10 to about 100 mg per kg (mg/kg) of body weight when thecompound is administered orally or intraperitoneally (IP). Whenadministered intravenously, the dosage should be approximately 50% ofthe oral or IP dosage to achieve the same level of the drug in the bloodstream. In this regard, it should be noted that a lethal dose may be inthe range of about 100 to about 400 mg/kg in small mammals and theadministered dose should thus be well below that level. The describeddosage should also be appropriate for prevention of human plateletaggregation or blood thinning. As is known to those skilled in the art,therapeutic doses expressed in terms of amounts per kilogram of bodyweight or surface area may be extrapolated from mammal to mammal,including to human beings.

The compounds of the present invention are particularly useful whenapplied topically to a wound. In this regard, the compounds may beincorporated into conventional ointment, lotion or aerosol formulations.Ointments may be prepared by incorporating 0.1 to 10% of the compound asan oil or sodium salt into an ointment base containing emulsifyingagents such as stearic acid, triethanolamine and/or cetyl alcohol. theformulation may also include ingredients such as glycerol, water andpreservatives as required.

Water based lotions may contain the compounds of the invention as an oilor as a sodium salt in amounts ranging from 0.1 to 5.0% by volume. Suchlotions may contain glycerine and/or bentonite as suspending agents asis well known in the art.

The compounds may also be incorporated into classical (one or two phase)or non-classical (aqueous emulsion) aerosol formulations. Suchformulations include the compounds and an appropriate propellant carrierin which the compounds are dissolved or dispensed. In the classical formthe active ingredients are generally used as an oil dispersion or insolution in an organic solvent such as ethanol. In the non-classicalform the active ingredient is dissolved in water. In each case theconcentration of the active ingredient in the carrier may be about 0.1to 10% by weight or volume.

The compounds of the invention may also be highly useful in the form ofgauze bandages which have been coated or impregnated with a solution ordispersion of the active material.

Of particular advantage is the fact that the cis-unsaturated straightchain fatty moiety compounds described above function pharmacologicallyat the site of inhibitory action for the arachidonate cascade, andpreferentially effect stimulus-induced mobilization of arachidonate.Inhibition of PLA₂ depresses the production of both prostaglandins andleukotrienes in stimulated or inflamed cells. Importantly, the polymersdescribed above have a much more pronounced effect on stimulus-induced,than on controlled release of arachidonate indicating a selective effecton the former. Moreover, when phospholipids are peroxidized, the polymercompounds described above are capable of inhibiting the degradation ofsuch lipids by lysosomal phospholipase C, indicating that thesecompounds can protect already damaged (oxidized) membranes.

Thus, multiple actions are responsible for the anti-inflammatoryactivity of the fatty moiety compounds of the invention, and on thebasis of inflammatory models, it is evident that these compounds caneffectively rival or replace both currently available steroids andNSAIAs in the treatment of inflammation, making the polymerizedcis-unsaturated straight chain fatty moiety compounds of the inventioncandidates for clinical application and usefulness in localized andsystemic injury and disease.

The fact that production of the prostaglandin, thromboxane, from freearachidonic acid is required for platelet function indicates that theunsaturated fatty moiety polymers described above should affect theplatelet aggregation release reaction.

PLA₂ is present at widely different levels in a variety of human cellsand fluids tested. Inflammation is associated with a significant rise inextracellular phospholipases, and the polymers of cis-unsaturated fattymoieties described above have the ability to inhibit these enzymes.

Phospholipases are released from pathogenic invading organisms and thepolymers described above act by inhibiting the action of thesemembrane-destructive enzymes produced by pathogens of bacterial,protozoal, viral, Rickettsial, helminthic and fungal origin. The actionof the compounds of the invention in preventing inflammation or tissueinjury is manifested by inhibition of PLA₂ at its source from infectingorganisms or inhibition by blocking host responses to infection orinjury.

In addition, there is evidence that tumor metastases or invasion isassociated with endogenous activity on the part of malignant cells andthe expression of phospholipases and their inhibition can play a role inthe control of differentiation and functional integrity as well as theprocesses of carcinogenesis and aging. In the latter case, the polymersof unsaturated fatty moieties described above prolong life in animalmodels and minimize membrane alterations in living organisms produced bycarcinogens (mutagens) and photo-oxidizers which are radiation-like intheir cell damaging activity.

The polymers of unsaturated fatty moieties described above, byprotecting lipid membranes and possessing anti-oxidant activity, arepotent anti-oxidants for preservation, not only of living cells andtissues, but their action makes them effective as a preservatives offood, tissue and chemical agents subject to oxidative injury. Forpurposes of protecting and preserving food subject to oxidation injury,the fatty moiety compounds of the invention may be used atconcentrations of approximately 0.1 to 100 μM. These molarities arecalculated as the molarity that would be obtained if the drug weredissolved in a weight of water which is the same as the weight of thefood stuff to be preserved. For example, in vitro, anti-oxidant and/oranti-phospholipase applications, concentrations of from about 0.1 toabout 500 μM should be effective.

The essence of the action of the polymers of cis-unsaturated straightchain fatty moieties described above is the role they play in resistinginjury and permitting repair to phospholipid/protein membranes. Theyalso play a protective role in the stabilization of proteins. Thesecompounds clearly are cytoprotective agents which protect the cellmembrane from toxic, pathogenic, or age mediated events in the cellularor supportive environment. In final analysis, the minimal dose of theunsaturated fatty moiety compounds of the invention depends uponempirical considerations for relief from phospholipase mediated injuryand/or inflammation or to accomplish the desired cytoprotectivefunction. The maximum dose depends principally on the necessity ofavoidance of undesired side-effects and lethal doses.

We claim:
 1. A compound having the formula ##STR53## wherein each Rgroup is a fatty moiety having from 16 to 20 carbon atoms and at least 1cis-unsaturated double bond, and Z is a C₁ to C₅ aliphatic organicradical.
 2. A compound as set forth in claim 1, wherein the R groups arethe same or different and each is ##STR54##
 3. A compound as set forthin claim 1, wherein Z is CH₂ and R is
 4. A compound having the formulawherein each R group is a fatty moiety having from 16 to 20 carbon atomsand at least 1 cis-unsaturated double bond, and Z is a C₁ to C₅aliphatic organic radical.
 5. A compound as set forth in claim 4,wherein the R groups are the same or different and each is ##STR55## 6.A compound as set forth in claim 4, wherein Z is CH₂ and R is